Inhibitors for urokinase receptor

ABSTRACT

The present invention concerns peptides as inhibitors of the binding of urokinase to the urokinase receptor. These peptides, which are preferably cyclic, are suitable as pharmaceutical agents for diseases that are mediated by urokinase and its receptor.

This application is a national stage entry of International ApplicationNo. PCT/EP98/02179, filed Apr. 14, 1998 designating the U.S., whichclaims the benefit of European Application No. 97 106 024.9, filed Apr.11, 1997.

The present invention concerns peptides as inhibitors of the binding ofurokinase to the urokinase receptor. These peptides which are preferablycyclic are suitable as pharmaceutical agents for diseases which aremediated by urokinase and its receptor.

The serine protease uPA (urokinase-type plasminogen activator) isresponsible for various physiological and pathological processes such asthe proteolytic degradation of extracellular matrix material which isnecessary for the invasiveness and migration of cells and for tissueremodelling. uPA binds with high affinity (K_(D)=10⁻¹⁰−10⁻⁹ M) to themembrane-based uPA receptor (uPAR) on the cell surface.

The binding of uPA to its receptor is involved in many invasivebiological processes such as the metastatic spread of malignant tumours,trophoplast implantation, inflammation and angiogenesis. Henceantagonists of uPA are able to inhibit the invasiveness, metastaticspread and angiogenesis of tumours. uPA antagonists can be used asagents for the treatment of invasive and metastasising cancer diseasesin which uPA and uPAR occur at the invasive foci of tumours (Dano etal., The receptor for urokinase plasminogen activator: Stromal cellinvolvement in extracellular proteolysis during cancer invasion, in:Proteolysis and Protein Turnover, Barrett, A. J. and Bond, J., Editor,Portland Press, London, 1994, 239) e.g. in cancers of the breast, lung,intestine and ovaries. In addition uPA antagonists can also be used forother purposes in which it is necessary to inhibit the proteolyticactivation of plasminogen, for example to treat diseases such asarthritis, inflammation, osteoporosis, retinopathies and forcontraception.

The uPA receptor is described in WO 90/12091 and in the publications byPloug et al., J. Biol. Chem. 268 (1993), 17539 and Ronne et al., J.Immunol. Methods 167 (1994), 91.

uPA is synthesized as a single chain molecule (pro-uPA) and is convertedenzymatically into an active two-chain uPA. The uPA molecule is composedof three structurally independent domains, the N-terminal growthfactor-like domain (GFD, uPA 1-46), a kringle structure domain (uPA45-135) and the serine protease domain (uPA 159-411). GFD and thekringle domain together form the so-called aminoterminal fragment of uPA(ATF, uPA 1-135) which is produced by further proteolytic cleavage oftwo-chain uPA. ATF binds to the uPA receptor with a similar affinity asuPA.

The receptor-binding region of uPA spans the region of the amino acids12 to 32 since a peptide which contains the amino acid residues 12 to 32of uPA (in which case cysteine is replaced by alanine in position 19)competes with ATF for binding to the uPA receptor (Appella et al., J.Biol. Chem. 262 (1987), 4437-4440). In this publication it was alsoshown that this peptide also has an affinity for the uPA receptor aftercyclization by bridging the two cysteine residues at positions 12 and32. In an alternative approach Goodson et al., (Proc. Natl. Acad. USA 91(1994), 7129-7133) identified antagonistic uPA peptides for the uPAR byscreening a bacteriophage peptide library. These peptides had noapparent sequence homology to the natural uPAR-binding sequence of uPA.

Further investigations of the uPAR-binding region of uPA are describedin recent publications (Rettenberger et al., Biol. Chem. Hoppe-Seyler376 (1995), 587-594); Magdolen et al., Eur. J. Biochem. 237 (1996),743-751; Goretzki et al., Fibrinolysis and Proteolysis 11 (1997),11-19). The residues Cys19, Lys23, Tyr24, Phe25, Ile28, Trp30 and Cys31were identified as important determinants for a uPA/uPAR interaction. Inthese investigations a uPA peptide having the amino acids 16 to 32 ofuPA was identified as the most effective inhibitor.

Magdolen et al., (1996) supra analysed the uPAR binding region of theuPA molecule using a peptide having the amino acids 14 to 32 of uPA andpeptides derived therefrom. However, these peptides and also peptidesused by other research groups (cf. e.g. Appella et al., (1987) supra)have a relatively low affinity for uPAR.

WO-A-94/22646 discloses linear peptides with a length of 6 to 18 aminoacids which are derived from the region of the amino acids 14 to 33 ofuPA. It is described that short peptides derived from uPA (uPA 21-29 anduPA 21-26) are able to influence the growth of keratinocytes. AlthoughWO-A-94/22646 makes reference to a potential use of the claimed peptidesto block the uPA/uPAR interaction, no data or information whatsoever areshown on such binding studies. Moreover, the peptides uPA 21-29 and uPA21-26 which are said to be preferred linear peptides do not contain theminimal uPAR binding region of linear uPA peptides which comprises thesequence region of amino acids 19 to 31. Hence the influence of thegrowth of keratinocytes by these short peptides is very probably not dueto a uPA/uPAR interaction.

However, a disadvantage of the previously known uPA peptide inhibitorsis that their affinity of binding to the uPA receptor is relatively lowand inadequate for a therapeutic application. Thus there is a great needfor new uPA peptide antagonists which have a higher affinity for thereceptor.

In quantitative investigations it was surprisingly found that the linearpeptide uPA (19-31) (SEQ ID NO: 3), cyclic derivatives of this peptideand sequence-modified peptides from this uPA region have a considerablyimproved affinity of binding to the uPA receptor.

Experimental data demonstrate that the peptides according to theinvention can be used as uPA antagonists which bind with high affinityto the uPAR. Cyclic peptides are particularly preferred which arecharacterized by bridges, especially disulfide bridges, which do notoccur in the native uPA molecule.

Hence the present invention concerns peptides having the generalstructural formula (I):

in which

-   -   X²¹ to X³⁰ each denotes an aminocarboxylic acid,    -   preferably an α-aminocarboxylic acid and X²¹ and X²⁹ are bridged        together,    -   Y is a spacer    -   m and n are each independently 0 or 1,    -   and the monomeric building blocks are linked by —NR¹CO— or        —CONR¹— bonds where R¹ in each case independently denotes        hydrogen, methyl or ethyl, and pharmaceutically compatible salts        and derivatives thereof.

The monomeric building blocks X²¹ to X³⁰ have preferably the followingmeanings:

-   -   X²¹ and X²⁹ are α-aminocarboxylic acid building blocks which can        be bridged together and they particularly preferably have an SH        side chain, in particular a cysteine side chain or a        structurally related side chain e.g. a penicillamine side chain.        Alternatively X²¹ and X²⁹ can also be two α-aminocarboxylic acid        residues linked by a thioether group e.g. a lanthionine group.    -   X²² and X²⁷ are each independently α-aminocarboxylic acids with        an aliphatic side chain, preferably an aliphatic hydrophilic        side chain and in particular an amide side chain such as        asparagine or glutamine, in particular asparagine.    -   X²³ is an α-aminocarboxylic acid with a basic side chain e.g.        lysine, ornithine or arginine or with an aliphatic hydrophilic        side chain e.g. with an amide side chain such as glutamine or        asparagine. X²³ is particularly preferably lysine.    -   X²⁴ to X²⁵ are each independently α-aminocarboxylic acids with        an aromatic side chain such as tyrosine, phenylalanine or        tryptophan. X²⁴ is particularly preferably tyrosine and X²⁵ is        phenylalanine.    -   X²⁶ is an α-aminocarboxylic acid with an aliphatic side chain,        preferably with an aliphatic hydrophilic side chain such as        hydroxyvaline, homoserine, serine or threonine, in particular        serine. However, X²⁶ can also have an aliphatic hydrophobic side        chain such as alanine.    -   X²⁸ is an α-aminocarboxylic acid with an aliphatic side chain,        preferably with an aliphatic hydrophobic side chain such as        valine, norvaline, norleucine, isoleucine, leucine or alanine.        X²⁸ is particularly preferably isoleucine.    -   X³⁰—if present—is an α-aminocarboxylic acid with an aromatic        side chain, preferably with a tryptophan side chain. The        tryptophan side chain can be optionally modified for example by        reduction.

The peptides according to the invention are preferably derived from theuPA sequence and contain at least 2 and particularly preferably at least3, for example 4 amino acid residues which also occur at correspondingpositions in the native uPA sequence. At least two of the amino acidresidues X²², X²³, X²⁴, X²⁵, X²⁶, X²⁸ and X³⁰ particularly preferablyhave a side chain which is identical to an amino acid at the sameposition in the native uPA sequence. Most preferably at least 2 of theamino acid residues X²⁴, X²⁵, X²⁸ and—if present—X³⁰ have the same sidechain as in the native uPA sequence.

Y is a spacer group e.g. a peptidic spacer group composed of one orseveral amino acids e.g. poly-Lys or another spacer group e.g. apolyethylene glycol group. The peptide can be coupled to carriersubstances via the group Y.

Hence a further subject matter of the present invention are cyclicpeptides with a nine-membered ring of which at least two, preferably atleast 3 and particularly preferably at least 4 of the amino acidsforming the ring have a sequence from the uPA region 22 to 28.

In addition to peptides having the structural formula (I),pharmaceutically compatible salts and derivatives thereof are alsosuitable as uPA antagonists. Suitable derivatives are in particularcompounds in which the reactive groups of the side chain or/and of theN-terminus or C-terminus e.g. amino or carboxylic acid groups have beenmodified. Examples of such modifications are acylation e.g. anacetylation of amino groups or/and an amidation or esterification ofcarboxylic acid groups.

Natural amino acids or enantiomers thereof or non-naturally-occurringamino acids such as γ-aminobutyric acid, β-alanine can be used as theaminocarboxylic acids that the building blocks for the peptidesaccording to the invention.

The monomeric building blocks are linked by acid amide bonds NR¹CO orCONR¹ i.e. the direction of the peptide sequence can also be reversed(retropeptides). As in native polypeptides, R¹ can denote hydrogen. Onthe other hand, R¹ can also denote an alkyl residue e.g. methyl or ethyland in particular methyl since N-alkylation of the amide bond often hasa major influence on the activity (cf. e.g. Levian-Teitelbaum et al.,Biopolymers 28 (1989), 51-64).

The α-aminocarboxylic acids can also be used as monomeric buildingblocks in the form of L-enantiomers or/and D-enantiomers. The spatialstructure of the peptides according to the invention can be modified bychanging the chirality which can also influence the activity.Retro-inverso peptides are particularly preferred i.e. peptides whichare present in a reversed sequence direction and contain D-amino acidsas monomeric building blocks. In these retro-inverso structures thefunctional side chains have a similar spatial orientation to those inthe native peptide sequence, but their biological degradation can beimpaired due to the presence of D-amino acids and they therefore haveadvantages as drugs (cf. for example Wermuth et al., J. Am. Chem. Soc.119 (1997), 1328-1335 and references cited therein).

The peptides according to the invention are preferably cyclic compoundsin which in particular the monomeric building blocks X²¹ and X²⁹ arebridged together. This bridging can for example utilize the side chainsof the respective α-aminocarboxylic acid residues in which case bridgingby means of disulfide bonds e.g. between two cysteine residues isparticularly preferred. Other types of cyclization between amino acidside chains are, however, also possible e.g. amide bonds between anamino acid with an amino side group e.g. ornithine or Lys and an aminoacid with a carboxylic acid side group such as Asp or Glu. In additionthe disulfide bridge can also be replaced by an alkylene bridge in orderto increase the chemical stability. In addition an amino acid side chainmay also be linked to the peptide backbone e.g. an ω-amino side groupmay be linked to the C-terminal end or a carboxylic acid side group maybe linked to the N-terminal end. A linkage of the N-terminus andC-terminus is also possible.

It is particularly preferred when at least one of the amino acids X²¹,X²⁷, X²⁹ and X³⁰ is a D-amino acid. At least one of the amino acids X²¹to X³⁰ is particularly preferably a D-amino acid e.g. D-cysteine.

Instead of the disulfide bridge it is also possible to use so-calledturn mimetics (Haubner et al., J. Am. Chem. Soc. 118 (1996), 7884-7891)or sugar amino acids (Graf von Rödern et al., J. Am. Chem. Soc. 118(1996), 10156-10167).

The peptides according to the invention can be obtained by chemicalsynthesis as elucidated in the examples. Alternatively the peptidesaccording to the invention can also be components of recombinantpolypeptides.

Yet a further subject matter of the present invention are peptides whichare derived from the linear peptide uPA (19 to 31) and cyclicderivatives thereof and carry D-amino acid residues at selectedpositions. Such peptides have the general structural formula (II):X¹—[X²]_(n)—[X³]_(m)—X⁴—K—Y—F—X⁵—X⁶—I—X⁷—W—[X⁸]_(r)  (II)in which

-   -   X¹ to X⁸ each denotes an aminocarboxylic acid preferably an        α-aminocarboxylic acid and X¹ and X⁷ or X¹ and X⁸ are optionally        bridged together,    -   n, m and r are each independently 0 or 1,    -   K is defined as X²³ and preferably denotes an α-amino-carboxylic        acid with a lysine side chain,    -   Y is defined as X²⁴ and preferably denotes an α-amino-carboxylic        acid with a tyrosine side chain,    -   F is defined as X²⁵ and preferably denotes an α-amino-carboxylic        acid with a phenylalanine side chain,    -   I is defined as X²⁸ and preferably denotes an α-amino-carboxylic        acid with an isoleucine side chain,    -   W is defined as X³⁰ and preferably denotes an α-amino-carboxylic        acid with a tryptophan side chain        and the monomeric building blocks are linked by —CONR¹— or        —NR¹CO— bonds where R¹ in each case independently denotes        hydrogen, methyl or ethyl and pharmaceutically compatible salts        and derivatives thereof and in which at least one of the amino        acid residues denotes X¹, X², X³, X⁶, I, X⁷, W and X⁸ denotes a        D-amino acid residue.

The monomeric building blocks X¹ to X⁸ preferably have the followingmeanings:

-   -   X¹ and—if present—X⁸ correspond to the meaning of X²¹ and X²⁹        and are e.g. α-aminocarboxylic acid building blocks with an SH        side chain, in particular with a cysteine side chain.    -   X²—if present—is an α-aminocarboxylic acid with an aliphatic and        uncharged side chain e.g. valine, leucine or isoleucine, in        particular valine.    -   X³ and X⁵ correspond to the meaning of X²⁶ and are e.g.        α-aminocarboxylic acids with an aliphatic hydrophilic side chain        such as serine or threonine, in particular serine.    -   X⁴ and X⁶ correspond to the meaning of X²² and X²⁷ and are e.g.        α-aminocarboxylic acids with an aliphatic hydrophilic side        chain, in particular an amide side chain such as asparagine or        glutamine, in particular asparagine.

If not bridged with X¹, X⁷ is preferably a basic α-aminocarboxylic acid,in particular histidine. If it is bridged with X¹, then X⁷ is anα-aminocarboxylic acid with an SH side group, in particular cysteine.

The present invention additionally concerns a pharmaceutical compositionwhich contains at least one peptide or polypeptide as defined above asthe active substance optionally together with common pharmaceuticalcarriers, auxiliary agents or diluents. The peptides or polypeptidesaccording to the invention are used especially to produce uPAantagonists which are suitable for treating diseases associated with theexpression of uPAR especially for treating tumours.

An additional subject matter of the present invention is the use ofpeptides derived from the uPA sequence and in particular of uPAantagonists such as the above-mentioned peptides and polypeptides toproduce targeting vehicles e.g. liposomes, viral vectors etc. foruPAR-expressing cells. The targeting can be used for diagnosticapplications to steer the transport of marker groups e.g. radioactive ornon-radioactive marker groups. On the other hand the targeting can befor therapeutic applications e.g. to transport pharmaceutical agents andfor example also to transport nucleic acids for gene therapy.

The pharmaceutical compositions according to the invention can bepresent in any form, for example as tablets, as coated tablets or in theform of solutions or suspensions in aqueous or non-aqueous solvents. Thepeptides are preferably administered orally or parenterally in a liquidor solid form. When they are administered in a liquid form, water ispreferably used as the carrier medium which optionally containsstabilizers, solubilizers or/and buffers that are usually used forinjection solutions. Such additives are for example tartrate or boratebuffer, ethanol, dimethyl sulfoxide, complexing agents such as EDTA,polymers such as liquid polyethylene oxide etc.

If they are administered in a solid form, then solid carrier substancescan be used such as starch, lactose, mannitol, methyl cellulose, talcum,highly dispersed silicon dioxide, high molecular fatty acids such asstearic acid, gelatin, agar, calcium phosphate, magnesium stearate,animal and vegetable fats or solid high molecular polymers such aspolyethylene glycols. The formulations can also contain flavourings andsweeteners if desired for oral administration.

The therapeutic compositions according to the invention can also bepresent in the form of complexes e.g. with cyclodextrins such asγ-cyclodextrin.

The administered dose depends on the age, state of health and weight ofthe patient, on the type and severity of the disease, on the type oftreatment, the frequency of the administration and the type of desiredeffect. The daily dose of the active compound is usually 0.1 to 50mg/kilogramme body weight. Normally 0.5 to 40 and preferably 1.0 to 20mg/kg/day in one or several doses are adequate to achieve the desiredeffects.

The invention is further illustrated by the examples described in thefollowing and the figures.

FIG. 1 shows the quantity-dependent inhibition of the binding of pro-uPAto a cell surface-associated uPAR by synthetic peptides;

FIG. 2 shows the competition of synthetic peptides with ATF for bindingto the uPAR;

FIG. 3A shows the structure of cyclo¹⁹⁻³¹ uPA 19-31 (right) (SEQ ID NO:3) compared to the structure of the corresponding domain from native uPA(SEQ ID NO: 2) and

FIG. 3B shows the structure of the cyclic peptide derivativecyclo^(21,29) [Cys21,29] uPA₂₁₋₃₀ (SEQ ID NO: 4).

FIG. 4 shows the inhibition of the uPA/uPAR interaction by syntheticpeptides and

FIG. 5 shows the inhibition of tumour growth in naked mice byadministration of synthetic peptides.

EXAMPLES 1. Methods

1.1 Solid Phase Peptide Synthesis

Linear peptides were synthesized on a 2-chlorotrityl resin (Barlos etal., Int. J. Pept. Protein Res. 37 (1991), 513 to 520) using an AppliedBiosystems Model 431 A peptide synthesizer or a multiple peptidesynthesizer model Syro II (MultiSynTech). Using the orthogonal Fmocstrategy (Carpino and Han, J. Org. Chem. 37 (1972), 3404-3409; Fieldsand Noble, Int. J. Peptide Protein Res. 35 (1990), 161-214) the aminoacid side chains were blocked with the protecting groups trityl (Asn,Cys, Gln and His), tert.-butyloxycarbonyl (Lys and Trp), tert.-butyl(Asp, Glu, Ser, Thr and Tyr), acetamidomethyl (Cys) and2,2,5,7,8-pentamethylchroman-6-sulfonyl or2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Arg). The couplingwas carried out at room temperature in dimethylformamide using athree-fold excess of2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumtetrafluoroborate/1-hydroxybenzotriazole/Fmoc-aminoacid with 2.5 equivalents of N-ethyldiiso-propylamine inN-methyl-pyrrolidone. The Fmoc group was removed by sequential treatmentof the resins with an excess of 40% or 20% piperidine indimethylformamide. The cleavage of the peptides and removal of the sidechain protecting groups was carried out simultaneously by treatment with82.5% trifluoroacetic acid/5% phenol/2.5% ethane dithiol/5%thioanisol/5% H₂O (0° C./1h; room temperature/1h). In the case of Arggroups protected with 2,2,5,5,7,8-pentamethylchroman-6-sulfonyl, thepeptides were incubated for an additional 12 h at room temperature. Thecrude peptides were precipitated at −30° C. with diethyl ether,dissolved in methanol, precipitated as previously described, dissolvedin tert.-butanol and lyophilized. Peptides containing tryptophan wereadditionally treated for 2 h with 5% acetic acid before thelyophilization.

The peptides were purified by HPLC using a reversed phase C18 column(Nucleosil 1005-C18) or a YMC pack ODS column. They were cyclized byforming a disulfide bridge between the cysteine residues. The oxidationrequired for this was carried out by taking 0.1 to 0.3 mg/ml of thepurified linear peptides up in 80% water and 20% DMSO (vol/vol) andremoving the solvent under reduced pressure after 10 h. The cyclicpeptides were again purified by HPLC as described above.

1.2 Mass Spectroscopy and Amino Acid Analysis

The purified and desalted peptides were analysed on a HPLC system 140 B(Applied Biosystems, Foster City, USA). The UV absorbance was measuredwith a UVIS 200 detector (Linear Instruments, Reno, USA) at 206 nm. Thechromatography was carried out on an Aquapore 3 μ(Applied Biosystems,Foster City, USA) reversed phase column (1 mm×50 mm) at a flow rate of20 μl/min. The solvent system was 0.1% TFA in water (A) and 0.1% TFA inacetonitrile (B). The HPLC system was coupled to an atmospheric pressureionisation source which was connected to a tandem quadrupole instrumentAPI III (Sciex, Perkin Elmer, Thornhill, Canada).

The quadrupole M/Z scale was calibrated with the ammonium additionproducts of polypropylene glycol. The average mass values werecalculated from the M/Z peaks in the charge distribution profiles of themultiple charged ions (Covey et al., Rapid Commun. Mass Spectrom. 2(1988), 249-256; Fenn et al., Science 246 (1989), 64-71).

The amino acid analysis was carried out according to the ninhydrinmethod using the analytical system 6300 (Beckman Instruments, Fullerton,USA) after hydrolysing the peptides by the TFA-HCl vapour phase methodwhich allows a quantitative determination of the peptide concentration(Tsugita et al., J. Biochem. 102 (1987), 1593-1597).

1.3 Flow Cytometry

The ability of the synthetic peptides to inhibit the uPA/uPARinteraction was determined by means of flow cytometry on a FACScan flowcytometer (Becton-Dickinson, Heidelberg, Germany) using the humanpromyeloid cell line U937 as a source of cellular native uPAR(Chuchulowski et al., Fibrinolysis 6, Suppl. 4 (1992), 95-102; Magdolenet al., (1996), supra). The U937 cells were stimulated with 1 mMphorbol-12-myristate-13-acetate (PMA) for 48 h. After stimulation withPMA the U937 cells expressed considerable amounts of cellsurface-associated uPAR.

The stimulated cells were treated for 1 min at room temperature with 50mM glycine HCl, 0.1 NaCl, pH 3.6 in order to dissociate endogenousreceptor-bound uPA. Subsequently the acidic buffer was neutralized with0.5 M HEPES-100 mM NaCl, pH 7.5. The cells were then immediately washedtwice with PBS/0.1% bovine serum albumin (BSA) and centrifuged for 10min at room temperature and 300 x g. The cells were resuspended inPBS/0.1% BSA, adjusted to a concentration of 10⁶ cells per ml andsimultaneously incubated for 45 minutes at room temperature with 16 ngFITC-conjugated pro-uPA and various amounts of the synthetic peptides.Before the analysis, propidium iodide, a fluorescent dye whichspecifically binds double-stranded DNA, was added to each sample inorder to determine the viability of the analysed U937 cells. Damaged,propidium iodide-labelled cells were excluded from the analysis.

1.4 Solid Phase uPAR/uPA Binding Test

In addition to the flow cytometric analyses, a solid phase ATF-ligandbinding test was carried out in order to examine the interactions ofsynthetic peptides with the uPAR. For this microtitre plates were coatedwith recombinant human uPAR from CHO cells (Wilhelm et al., FEBS Lett.337 (1994), 131-134; Magdolen et al., Electrophoresis 16 (1995),813-816) and the remaining protein-binding sites were saturated with 2%BSA (weight/vol). After incubation with the samples (0.6 ng ATF togetherwith 15 μg synthetic peptide per ml) and several wash steps, the amountof ATF which had bound to the uPAR immobilized on the microtitre platewas determined using a biotinylated monoclonal mouse antibody againstthe kringle domain of ATF (No. 377, American Diagnostics, Greenwich,Conn., USA) and subsequent addition of avidin-peroxidase conjugate and3,3′,5,5′-tetramethylbenzidine/H₂O as a substrate for the peroxidase.The presence of synthetic peptides which compete with the ATF binding touPAR reduces the conversion of the chromogenic substrate.

2. Results

2.1 Determination of the uPAR Binding Capacity of Synthetic Peptides byQuantitative Flow Cytometric Analysis

A comparison was made of the inhibitory capacity of the peptidesuPA₁₂₋₃₂ [C19A] (Appella et al., (1987), supra) the so-called clone20-peptide AEPMPHSLNFSQYLWYT (SEQ ID NO:1) (Goodson et al., (1994),supra) which was identified as the most effective peptide from a phagepeptide library and of the synthetic peptide uPA₁₆₋₃₂ derived from thewild-type uPA sequence.

For this the purified peptides were analysed by mass spectroscopy,quantified by amino acid analysis and then tested by flow cytometryaccording to the method described in 1.3 for their ability to inhibitthe binding of fluorescent-labelled pro-uPA to the uPA receptor on U937cells. It was found that pro-uPA is displaced in a dose-dependent mannerfrom the cell surface-associated uPAR by all three synthetic peptides(FIG. 1). An approximately 15,000 to 12,000 molar excess of uPA₁₂₋₃₂[C19A] or clone 20 peptide resulted in a 50% inhibition of the bindingof uPA. The peptide uPA₁₆₋₃₂ exhibited a 4- to 5-fold higher affinity touPAR compared to the two other peptides: an approximately 3,000-foldmolar excess is sufficient to achieve a 50% inhibition.

Furthermore it was found that the linear peptide uPA₁₉₋₃₁ surprisinglyhas an IC50 value of ca. 0.8 μM whereas the IC50 value for uPA₁₆₋₃₂ isca. 3.2 μM.

2.2 Determination of the uPAR Binding Capacity of Synthetic Peptides ina Microtitre Plate Solid Phase Ligand Binding Test

A series of peptides with variable sequence regions from the receptorbinding region of uPA were synthesized and were increasingly shortenedat the amino terminus starting with uPA₁₀₋₃₂. The microtitre plate solidphase binding test described in 1.4 was used to determine the inhibitorycapacity of these peptides. The results of this test are shown in FIG.2.

It can be seen in FIG. 2A that the peptides uPA₁₀₋₃₂, uPA₁₂₋₃₁, uPA₁₄₋₃₂and uPA₁₆₋₃₂ effectively inhibit the binding of ATF to uPAR. Thepeptides uPA₁₇₋₃₂ and uPA₁₈₋₃₄ have considerably reduced uPAR bindingcapacities. The peptide uPA₂₀₋₃₄ does not bind at all to the uPAR. In afurther experiment the binding capacity of the peptides uPA₁₉₋₃₁ (SEQ IDNO: 3), uPA₁₈₋₃₀, uPA₂₀₋₃₂ and uPA₂₀₋₃₀ was tested. The result of thisexperiment is shown in FIG. 2B. Surprisingly it was found that uPA₁₉₋₃₁(SEQ ID NO: 3) binds to the uPAR with higher affinity than the longerpeptide uPA₁₆₋₃₂. The other tested linear peptides had no significantbinding capacity.

The cyclic peptide cyclo¹⁹⁻³¹uPA₁₉₋₃₁ (SEQ ID NO: 3) which contains anintramolecular disulfide bond between the cysteine residues at positions19 and 31 was surprising still able to inhibit the binding of uPA to theuPA receptor. Furthermore the binding activity of cyclo¹⁹⁻³¹uPA₁₉₋₃₁(SEQ ID NO: 3) was significantly more stable after long storage inaqueous solution or repeated freeze/thaw cycles then that of the linearpeptide uPA₁₉₋₃₁ (SEQ ID NO: 3).

2.3 Systematic replacement of L-amino acids by D-amino acids inchemically synthesized linear and cyclic peptides from the regionuPA₁₉₋₃₁ (SEQ ID NO: 3)

The uPAR binding capacity of synthetic linear and cyclic peptides fromthe region uPA₁₉₋₃₁ (SEQ ID NO: 3) was determined by in each casereplacing one L-amino acid by the corresponding D-amino acid. Theresults of this experiment are shown in the following table 1.

D-amino acid Peptide structure Inhibition Trp30 [D-Trp³⁰]uPA₁₉₋₃₁ (SEQID NO: 3) ++ Trp30 cyclo[D-Trp³⁰]uPA₁₉₋₃₁(SEQ ID NO: 3) + His29[D-His²⁹]uPA₁₉₋₃₁(SEQ ID NO: 3) ++ His29 cyclo[D-His²⁹]uPA₁₉₋₃₁(SEQ IDNO: 3) + Asn27 [D-Asn²⁷]uPA₁₉₋₃₁(SEQ ID NO: 3) ++ Asn27cyclo[D-Asn²⁷]uPA₁₉₋₃₁(SEQ ID NO: 3) ++ Ser21 [D-Ser²¹]uPA₁₉₋₃₁(SEQ IDNO: 3) ++ Ser21 cyclo[D-Ser²¹]uPA₁₉₋₃₁(SEQ ID NO: 3) ++ Val20[D-Val²⁰]uPA₁₉₋₃₁(SEQ ID NO: 3) ++ Val20 cyclo[D-Val²⁰]uPA₁₉₋₃₁(SEQ IDNO: 3) + Cys19 [D-Cys¹⁹]uPA₁₉₋₃₁(SEQ ID NO: 3) +++ Cys19cyclo[D-Cys¹⁹]uPA₁₉₋₃₁(SEQ ID NO: 3) +++ cyclo19-31cyclo[19-31]uPA₁₉₋₃₁(SEQ ID NO: 3) +++

It can be seen from this table that the introduction of D-amino acids atpositions Cys19, Val20, Ser21, Asn27, His29 and Trp30 in the linear aswell as in the cyclic peptides is possible without loss of theinhibitory effect. Moreover it was found that in the case of the linearpeptides the inhibitory effect is not lost by introducing D-amino acidsat positions Ile28 and Cys31.

2.4 Synthesis of Modified Cyclic uPA Peptides

Using cyclo19,31uPA₁₉₋₃₁ (SEQ ID NO: 3) as the lead structure, a cyclicpeptide was prepared in which certain amino acids were deleted and/orsubstituted by other amino acids. The structure of this new syntheticpeptide variant cyclo^(21,29)[Cys21,29]uPA₂₁₋₃₀ (SEQ ID NO: 4) is shownin FIG. 3. In contrast to the synthetic method stated in 1.1 thispeptide was prepared on a trityl chloride polystyrene resin.

FIG. 4 shows the inhibitory effect of this synthetic peptide variantcompared to cyclo^(19,31)uPA₁₉₋₃₁ (SEQ ID NO: 3) andcyclo^(19,31)[D-Cys19]uPA₁₉₋₃₁ (SEQ ID NO: 3).

2.5 In vivo Effect

6×10⁶ human breast cancer cells MDA-MB-231 (Price et al., Cancer Res. 50(1990), 717-721) in a total volume of 300 μl were injected into theright side of 4-6 week old Balbc/3 naked mice. Before injection thecancer cells were mixed with 200 μg of the cyclic uPA peptidescyclo^(19,31)uPA₁₉₋₃₁ (SEQ ID NO: 3) and Cyclo^(21,29)[Cys21,29]uPA₂₁₋₃₀(SEQ ID NO: 4) in PBS, pH 7.4. Subsequently the mice were treated twiceweekly intraperitoneally with the respective peptide at a dose of 10mg/kg body weight (injection volume 300 μl). The volume of the primarytumours which occurred in the mice in cm³ was determined after 1, 2, 3and 5 weeks by measuring the two largest diameters. The control micewere administered PBS pH 7.4. Each group was composed of 5 mice. Theresults for the peptide cyclo^(19,31)uPA₁₉₋₃₁ (SEQ ID NO: 3) are shownin Tab. 2.

TABLE 2 Week Control uPA peptide 1 0 0 2 0.34 ± 0.3 0.086 ± 0.047 3 0.71± 0.5 0.303 ± 0.129 5  2.33 ± 0.32  0.62 ± 0.21* *p = 0.02

The volume of the primary tumour after a five week treatment is shown inFIG. 5. It can be seen that the administration of both peptides led to asignificant reduction of the tumour growth in vivo.

1. A peptide comprising monomeric building blocks and having the general structural formula (I):

in which X²¹ to X³⁰ each denotes an aminocarboxylic acid and X²¹ and X²⁹ are bridged together, Y is a spacer group that can couple the peptide to carrier substances n and m are each independently 0 or 1, and the monomeric building blocks are linked by —NR¹CO— or —CONR¹— bonds where R¹ in each case independently denotes hydrogen, methyl or ethyl, and wherein the amino acid residues X²¹-X³⁰ each independently have one of the following meanings: (i) X²¹ and X²⁹ are each independently an aminocarboxylic acid residue with an SH side chain or X²¹ and X²⁹ are together two aminocarboxylic acid residues which are bridged by a thioether bond; (ii) X²² and X²⁷ are each independently an aminocarboxylic acid residue with an aliphatic side chain; (iii) X²³ is an aminocarboxylic acid residue with a basic or an aliphatic hydrophilic side chain; (iv) X²⁴, X²⁵ and X³⁰ are each independently an aminocarboxylic acid residue with an aromatic side chain, (v) X²⁶ is an aminocarboxylic acid residue with an aliphatic side chain, and (vi) X²⁸ is an aminocarboxylic acid residue with an aliphatic side chain; and a pharmaceutically compatible salt or derivative thereof.
 2. A peptide as claimed in claim 1, wherein (i) X²⁴ has a tyrosine side chain; (ii) X²⁵ has a phenylalanine side chain; (iii) X²⁸ has an alanine, leucine or isoleucine side chain, and (iv) X³⁰ has an optionally modified tryptophan side chain.
 3. A peptide as claimed in claim 1, wherein at least 2 of the amino acid residues X²², X^(23, X) ²⁴, X²⁵, X²⁶, X²⁷, X²⁸ and X³⁰ have an identical side chain to an amino acid at the corresponding X²², X²³, X²⁴, X²⁵, X²⁶, X²⁷, X²⁸ and X³⁰ position in a native uPA sequence.
 4. A peptide as claimed in claim 3, wherein at least 2 of the amino acid residues X²⁴, X²⁵, X²⁸ and X³⁰ have the same side chain as an amino acid at the corresponding X²⁴, X²⁵, X²⁸ and X³⁰ position in the native uPA sequence.
 5. A peptide as claimed in claim 1, wherein X²¹ and X²⁹ are bridged via side chains of amino-carboxylic acid residues.
 6. A peptide as claimed in claim 5, wherein X²¹ and X²⁹ are bridged by means of disulfide bonds.
 7. Pharmaceutical composition suitable for inhibiting the binding of urokinase to a urokinase receptor comprising at least one peptide or polypeptide as claimed in claim 1 as an active substance and at least one pharmaceutically acceptable carrier, auxiliary agent or diluent.
 8. The peptide of claim 1, wherein the side chains of X²² and X²⁷ are aliphatic hydrophilic side chains.
 9. The peptide of claim 1, wherein the side chains of X²² and X²⁷ are amide side chains.
 10. The peptide of claim 1, wherein the side chain of X²⁶ is an aliphatic hydrophilic side chain.
 11. The peptide of claim 1, wherein the side chain of X²⁶ is a hydroxy side chain.
 12. The peptide of claim 1, wherein the side chain of X²⁸ is an aliphatic hydrophobic side chain.
 13. The peptide of claim 2, wherein the side chain of X²⁸ an isoleucine side chain.
 14. A polypeptide comprising at least two peptides wherein at least one of said peptides comprises monomeric building blocks and has the general structural formula (I):

in which X²¹ to X³⁰ each denotes an aminocarboxylic acid and X²¹ and X²⁹ are bridged together, Y is a spacer group that can couple the peptide to carrier substances n and m are each independently 0 or 1, and the monomeric building blocks are linked by —NR¹CO— or —CONR¹— bonds where R¹ in each case independently denotes hydrogen, methyl or ethyl, and wherein the amino acid residues X²¹-X³⁰ each independently have one of the following meanings: (i) X²¹ and X²⁹ are each aminocarboxylic acid residues with an SH side chain or 2 aminocarboxylic acid residues which are bridged by a thioether bond; (ii) X²² and X²⁷ are each independently aminocarboxylic acid residues with an aliphatic side chain; (iii) X²³ is an aminocarboxylic acid residue with a basic or an aliphatic hydrophilic side chain; (iv) X²⁴, X²⁵ and X³⁰ are each independently aminocarboxylic acid residues with an aromatic side chain, (v) X²⁶ is an aminocarboxylic acid residue with an aliphatic side chain, and (vi) X²⁸ is an aminocarboxylic acid residue with an aliphatic side chain; or a pharmaceutically compatible salt and derivative thereof.
 15. A polypeptide as claimed in claim 14, wherein (i) X²⁴ has a tyrosine side chain; (ii) X²⁵ has a phenylalanine side chain; (iii) X²⁸ has an alanine, leucine or isoleucine side chain, and (iv) X³⁰ has an optionally modified tryptophan side chain.
 16. A polypeptide as claimed in claim 14, wherein at least 2 of the amino acid residues X²², X²³, X²⁴, X²⁵, X²⁶, X²⁷, X²⁸ and X³⁰ identical side chain to an amino acid at the corresponding X²², X²³, X²⁴, X²⁵, X²⁶, X²⁷, X²⁸ and X³⁰ position in a native uPA sequence.
 17. A polypeptide as claimed in claim 14, wherein at least 2 of the amino acid residues X²⁴, X²⁵, X²⁸ and X³⁰ have the same side chain as an amino acid at the corresponding X²⁴, X²⁵, X²⁸ and X³⁰ position in the native uPA sequence.
 18. A polypeptide as claimed in claim 14, wherein X21 and X29 are bridged via side chains of amino-carboxylic acid residues.
 19. A polypeptide as claimed in claim 18, wherein X²¹ and X²⁹ are bridged by means of disulfide bonds.
 20. The polypeptide of claim 14, wherein the side chains of X²² and X²⁷ are aliphatic hydrophilic side chains.
 21. The polypeptide of claim 14, wherein the side chains of X²² and X²⁷ are amide side chains.
 22. The polypeptide of claim 14, wherein the side chain of X²⁶ is an aliphatic hydrophilic side chain.
 23. The polypeptide of claim 14, wherein the side chain of X²⁶ is a hydroxy side chain.
 24. The polypeptide of claim 14, wherein the side chain of X²⁸ is an aliphatic hydrophobic side chain.
 25. The polypeptide of claim 14, wherein the side chain of X²⁸ an isoleucine side chain.
 26. A polypeptide comprising monomeric building blocks and having the general structural formula (I):

in which X²¹ to X³⁰ each denotes an aminocarboxylic acid and X²¹ and X²⁹ are bridged together, X²² and X²⁷ are each independently aminocarboxylic acid residues with a side chain comprising at least one amino acid independently selected from the group consisting of asparagine and glutamine; X²³ is an aminocarboxylic acid residue with a basic or a hydrophilic side chain comprising at least one amino acid independently selected from the group consisting of asparagine and glutamine; X²⁶ is an aminocarboxylic acid residue with a side chain comprising at least one amino acid independently selected from the group consisting of hydroxyvaline, homoserine, serine, threonine, and alanine, X²⁸ is an aminocarboxylic acid residue with a side chain comprising at least one amino acid independently selected from the group consisting of valine, norvaline, norleucine, isoleucine, leucine, or alanine, X²⁹ is an aminocarboxylic acid residue with an aliphatic side chain, Y is a spacer group that can couple the peptide to carrier substances n and m are each independently 0 or 1, and the monomeric building blocks are linked by —NR¹CO— or —CONR¹— bonds where R¹ in each case independently denotes hydrogen, methyl or ethyl, and a pharmaceutically compatible salt or derivative thereof. 